Choroidal neovascularization (CNV) is a severe complication of age-related macular degeneration (AMD). Human genetic studies have recently revealed significant association of the very low-density lipoprotein receptor (VLDLR) and a wnt co-receptor, low-density lipoprotein receptor-related protein 6 (LRP6) genes with AMD in human patients. Our recent studies showed that VLDLR knockout mice develop typical CNV, and manifest most abnormalities of human AMD such as retinal inflammation, vascular leakage, and impaired cone ERG and cone degeneration. Wnts are a group of secreted, cystine-rich glycoproteins which bind to frizzled (Fz) receptors or to a co-receptor complex consisting of Fz and LRP5 or LRP6 (LRP5/6) and regulate expression of target genes, such as VEGF. The wnt signaling is known to mediate multiple biological functions including angiogenesis. However, the role of the wnt pathway in CNV has not been investigated. Our preliminary studies have provided the following evidence suggesting a pathogenic role of VLDLR and the wnt pathway in CNV: 1) LRP5/6 expression is significantly up-regulated in Vldlr-/- eyecups. 2) The down-stream effectors of the wnt pathway, glycogen synthase kinase-32 (GSK- 32) and 2-catenin, are both activated in Vldlr-/- eyecups. 3) VEGF expression is up-regulated in Vldlr-/- eyecups. 4) In cultured endothelial cells, down-regulation of VLDLR by siRNA activates the wnt signaling and VEGF over-expression. 5) DKK1, a specific inhibitor of the wnt pathway blocks the VEGF over- expression induced by the VLDLR siRNA and in the RPE of Vldlr-/- mice. The central hypothesis of this project is that VLDLR functions as a negative regulator of CNV, and the regulatory effect of VLDLR is through the wnt pathway. In this project, we will first determine if the RPE-derived VEGF over-expression in Vldlr-/- mice is essential for the development of CNV. We will generate VLDLR/VEGF double knockout (KO) mice by crossing the RPE-specific VEGF conditional KO mice with Vldlr-/- mice, and determine if the VEGF KO in the RPE attenuates CNV in Vldlr-/- mice. We will also investigate if the VEGF over-expression induced by VLDLR KO is through HIF-1 using RPE-specific HIF-11/VLDLR double KO mice. Second, we will test the hypothesis that the CNV in Vldlr-/- mice is mediated by the wnt pathway. The activation of the 2-catenin will be determined in primary RPE and retinal endothelial cells from Vldlr-/- mice. We will investigate if inhibition of the wnt pathway by DKK1 will prevent or alleviate CNV in Vldlr-/- mice. Further, constitutively active mutants of LRP5 and LRP6 will be expressed in the RPE of transgenic mice to determine if over-activation of the wnt pathway alone will induce CNV. We will determine whether LRP5 or LRP6 mediates the CNV in Vldlr-/- mice by generating LRP5/VLDLR double KO mice. Third, we will investigate how VLDLR regulates LRP5/6 gene expression. Two postulated mechanisms will be tested: 1) VLDLR and LRP5/6 may compete for binding with the same ligand. When VLDLR is deficient, more ligand could bind to LRP5/6 and induce the expression of the receptors. 2) VLDLR may interact with an intracellular signaling pathway, through which VLDLR down-regulates LRP5/6 gene transcription. We will delete the intracellular domain and the extracellular ligand-binding domain of VLDLR and determine if the ligand binding domain or the intracellular domain interacting with signaling pathways is essential for its regulatory effect on LRP5/6 expression. If the ligand binding domain is essential, we will identify the ligand binding to both VLDLR and LRP. On the other hand, if the intracellular domain is essential, we will identify the signaling pathway which could interact with VLDLR. The role of the VLDL-inducible factor-1 and DAB-1/Src kinase pathways will be investigated as the first candidates. This project will establish VLDLR as a new member of the wnt pathway and establish the roles of VLDLR and the wnt pathway in pathogenesis of CNV. These studies have potential to reveal a new pathogenic mechanism of CNV and a new drug target of future treatment of CNV in AMD. PUBLIC HEALTH RELEVANCE: Choroidal neovascularization is a severe complication of age-related macular degeneration and a major cause of blindness in aged population. This project aims to explore a novel pathogenic mechanism for choroidal neovascularization and to establish a new animal model of choroidal neovascularization.